A ribonuclease (RNase) bound to an endogenous DNA inhibitor has been isolated from Citrobacter sp. The formation of the enzyme-inhibitor (EI) complex is not an artifact of the isolation procedure. Separation of the enzyme and DNA inhibitor was accomplished by phenol extraction of the EI complex. The enzyme has been previously purified to homogeneity. Characterization of the isolated inhibitor revealed that it is an heterogenous array of DNA molecules of small (9-11S) average size. As the temperature is raised, there is a large increase in the absorbance of the DNA inhibitor, indicating the double-stranded nature of this extrachromasomal DNA. Studies performed on the EI complex demonstrate that the DNA, which resembles a miniplasmid, inhibits RNase activity by either a competitive (using Tris-HC1 buffer) or a mixed (using potassium phosphate buffer) mechanism. K is a strong positive function of ionic strength with the consequence that increases in buffer concentrations reverse the inhibition of RNase activity with concomitant increased hydrolysis of substrate. Spermidine will also reverse inhibition, but at much lower concentrations than expected for an effect mediated by ionic strength alone.